Polarographic cell systems have met with wide acclaim particularly in the medical field, providing for detection and concentration measurement of many desired analytes. Enzymes are commonly used in such systems, especially in those situations wherein the analyte itself is not polarographically active but where a reaction product formed or reactant consumed by an enzymatic reaction with the analyte is polarographically active.
For example, in medical applications, one common procedure is to measure glucose in the blood of a patient. Typically, blood samples are withdrawn from the patient for an in-line analysis for glucose concentration using a glucose oxidase electrode with a polarographic detector for detecting H.sub.2 O.sub.2 generated in accordance with the reaction: ##STR1##
The hydrogen peroxide generated by the reaction is measurable by a polarographic detector and, by appropriate calibration and calculation, glucose content in the sample can be accurately determined by the H.sub.2 O.sub.2 formed in the reaction.
Other areas in which analyte detection is required abound. For example, other biological detection systems, drug detection systems, chemical process systems, including fermentation systems and others, all require precise measurement of a variety of analytes. One specific example is now seen in the diet soft drink bottling industry where the artificial sweetener, aspartame (N-L-a-aspartyl-L-phenylalanine-methyl ester), commonly known by the brand name "Aspartame", has become the sweetening agent of choice.
Soft drink bottlers must determine the sweetener level present in a multiplicity of syrup or precursor diet soft drink lots to serve as an indication of the amount of water they must add to the syrup to obtain acceptable soft drink formulations as dictated by the syrup manufacturers. Additionally, once the desired dilution of the syrup has been made, the bottlers often check sweetener concentration to ensure compliance with desired sweetener concentration ranges.
One present method utilized to determine "aspartame" concentration levels is to chemically cleave the methyl ester functional moiety from the N-L-a-aspartyl-L-phenylalanine-methyl ester molecule via chymotrypsin enzymatic reaction. The reaction proceeds as follows: ##STR2## The amount of methanol produced can then be polarographically determined in known enzymatic analysis systems by use of the following equation, with the H.sub.2 O.sub.2 produced being polarographically measured ##STR3##
Polarographic determination of the H.sub.2 O.sub.2 produced can, for instance, be made in polarographic cells of the type disclosed in U.S. Pat. Nos. 3,979,274 and 4,073,713 (Newman), both patents being hereby incorporated by reference herein.
In accordance with these known polarographic cell structures, a laminated enzyme membrane is provided in which the innermost membrane of the laminate is located adjacent the working electrode of the polarographic detection circuit. Higher weight molecular species are prohibited from passing through this innermost membrane so that they will not contact the working electrode, while the polarographically active substance, H.sub.2 O.sub.2, can permeate the membrane and contact the electrode.
One obstacle which inhibits application of the Newman laminated enzyme membrane approach to methanol concentration measurement is that the CH.sub.2 O produced as a result of the alcohol oxidase (AOX) catalyzed oxidation of methanol interferes with accurate determination of the H.sub.2 O.sub.2 at the working electrode. Possibly, the CH.sub.2 O produced at the enzyme site competes with MeOH for reaction with the enzyme or the CH.sub.2 O itself produced may directly interfere with current measurement at the working electrode surface.
Undesirable instrument responses were found when H.sub.2 O.sub.2 polarographic detection methods were used in Newman type laminated enzyme membrane systems with alcohol oxidase used as the catalytic enzyme. These responses included drooping or ramping of current measurement plateaus as recorded on strip chart recorders used in conjunction with the measurement equipment, especially at higher MeOH concentrations and slow return of the analytical equipment to an acceptable baseline current after analyte sample injection.
Accordingly, there is a need in the assay field to provide improved, more accurate analysis in those measurement systems wherein formaldehyde exists as a possible interferant.
There is a more specific need to provide for improved polarographic detection in laminated enzyme membrane systems of the Newman type described above wherein CH.sub.2 O present or formed in the system interferes with accurate polarographic measurement at the transducer.